Name: measuring post-stroke cerebral edema, infarct zone and blood-brain barrier breakdown in a single set of rodent brain samples
Uploaded: 2020-10-23T13:30:00
Description: Watch this Scientific Journal Video about Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples at JoVE.com

We thank Maryna Kuscheriava, Maksym Kryvonosov, Daryna Yakumenko and Evgenia Goncharyk of the Department of Physiology, Faculty of Biology, Ecology, and Medicine, Oles Honchar, Dnipro University, Dnipro, Ukraine for their support and helpful contributions to our discussions. The data obtained are part of Ruslan Kuts&#8217;s PhD thesis.

The following procedures were conducted according to the recommendations of the Declaration of Helsinki and Tokyo and the Guidelines for the Use of Experimental Animals of the European Community. The experiments were also approved by the Animal Care Committee at the Ben-Gurion University of the Negev.
1. Preparing rats for the experimental procedure
<ol>
	<li>Select adult male Sprague-Dawley rats without overt pathology, each weighing between 300 and 350 g.</li>
	<li>Maintain all rats at roo...

The principal goal of the present protocol was to demonstrate consistent measurements of three main parameters of ischemic injury: IZ, BE and BBB permeability. Previous studies in this field have demonstrated the possibility of performing one or two of these parameters together in the same sample. Besides the cost reduction that this three-part method offers, it also provides a more desirable bioethical model that limits the number of animals that must be operated on and subsequently euthanized. As in all histological te...

One of the most common causes of morbidity and mortality worldwide is stroke. Globally, ischemic stroke represents 68% of all stroke cases, while in the United States ischemic stroke accounts for 87% of stroke cases1,2. It is estimated that the economic burden of stroke reaches $34 billion in the United States2 and &#8364;45 billion in the European Union3. Animal models of stroke are necessary to study its pathophysiology, develop new methods for evaluation, and propose new therapeutic options4.
Ischemic stroke occurs with occlusion of a major cerebral artery, usually the middle cerebral artery or one of its branches5. Thus, models of ischemic stroke have historically involved middle cerebral artery occlusion (MCAO)6,7,8,9,10,11,12. Following MCAO, neurological injury is most commonly assessed by measuring infarct zone (IZ) using a 2,3,5-triphenyltetrazolium chloride (TTC) staining method13, brain edema (BE) using drying or calculating hemispheric volumes14,15,16, and blood brain barrier (BBB) permeability by a spectrometry technique using Evans blue staining17,18,19.
The traditional MCAO method uses separate sets of brains for each of the three brain measurements. For a large sample size, this results in a significant number of euthanized animals, with added ethical and financial considerations. An alternative method to alleviate these costs would involve measurements of all three parameters in a single set of post-MCAO rodent brains.
Previous attempts have been made to measure combinations of parameters in the same brain sample. Simultaneous immunofluorescent staining methods20 as well as other molecular and biochemical analyses21 have been described after TTC staining in the same brain sample. We have previously calculated brain hemisphere volumes to assess brain edema and performed TTC staining to calculate infarct zone in the same brain set15.
In the present protocol, we present a modified MCAO technique that measures ischemic brain injury through determining IZ, BE, and BBB permeability in the same set of rodent brains. IZ is measured by TTC staining, BE is determined by calculating hemispheric volume, and BBB permeability is obtained by spectrometry methods19. In this protocol, we used a modified MCAO model, based on direct insertion and fixation of the monofilament catheter into the internal carotid artery (ICA) and further blocking of blood flow to the middle cerebral artery (MCA)22. This modified method shows a decreased rate of mortality and morbidity compared to the traditional MCAO method16,22.
This new approach provides a financially-sound and ethical model for measuring neurological injury after MCAO. This assessment of the main parameters of ischemic brain injury will help to comprehensively investigate its pathophysiology.

<table>
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			<td>2 mL Syringe</td>
			<td>Braun</td>
			<td>4606027V</td>
			<td></td>
		</tr>
		<tr>
			<td>2% chlorhexidine in 70% alcohol solution</td>
			<td>Sigma-Aldrich</td>
			<td>500 cc</td>
			<td>Provides general antisepsis of the skin in the operatory field</td>
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		<tr>
			<td>27 G Needle with Syringe</td>
			<td>Braun</td>
			<td>305620</td>
			<td></td>
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			<td>3-0 Silk sutures</td>
			<td>Henry Schein</td>
			<td>1007842</td>
			<td></td>
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			<td>4-0 Nylon suture</td>
			<td>4-00</td>
			<td></td>
			<td></td>
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			<td>Brain &#38; Tissue Matrices</td>
			<td>Sigma-Aldrich</td>
			<td>15013</td>
			<td></td>
		</tr>
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			<td>Cannula Venflon 22 G</td>
			<td>KD-FIX</td>
			<td>183603985447</td>
			<td></td>
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			<td>Centrifuge Sigma 2-16P</td>
			<td>Sigma-Aldrich</td>
			<td>Sigma 2-16P</td>
			<td></td>
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			<td>Compact Analytical Balances</td>
			<td>Sigma-Aldrich</td>
			<td>HR-AZ/HR-A</td>
			<td></td>
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			<td>Digital weighing scale</td>
			<td>Sigma-Aldrich</td>
			<td>Rs 4,000</td>
			<td></td>
		</tr>
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			<td>Dissecting scissors</td>
			<td>Sigma-Aldrich</td>
			<td>Z265969</td>
			<td></td>
		</tr>
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			<td>Eppendorf pipette</td>
			<td>Sigma-Aldrich</td>
			<td>Z683884</td>
			<td></td>
		</tr>
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			<td>Eppendorf tube</td>
			<td>Sigma-Aldrich</td>
			<td>EP0030119460</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence detector</td>
			<td>Tecan, M&#228;nnedorf Switzerland</td>
			<td>Model: Infinite 200 PRO multimode reader</td>
			<td>Optional.</td>
		</tr>
		<tr>
			<td>Fluorescence detector</td>
			<td>Molecular Devices LLC</td>
			<td>VWR cat. # 10822 512 SpectraMax Paradigm Multi Mode Microplate Reader Base Instrument</td>
			<td>Optional.</td>
		</tr>
		<tr>
			<td>Gauze sponges</td>
			<td>Fisher</td>
			<td>22-362-178</td>
			<td></td>
		</tr>
		<tr>
			<td>Heater with thermometer</td>
			<td>Heatingpad-1</td>
			<td>Model: HEATINGPAD-1/2</td>
			<td></td>
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		<tr>
			<td>Hemostatic microclips</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Horizon-XL</td>
			<td>Mennen Medical Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Infusion cuff</td>
			<td>ABN</td>
			<td>IC-500</td>
			<td></td>
		</tr>
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			<td>Micro forceps</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
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			<td>Micro scissors</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multiset</td>
			<td>Teva Medical</td>
			<td>998702</td>
			<td></td>
		</tr>
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			<td>Olympus BX 40 microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
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			<td>Operating forceps</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
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			<td>Operating scissors</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optical scanner</td>
			<td>Canon</td>
			<td>Cano Scan 4200F</td>
			<td>Resolution 3200 x 6400 dpi</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Sigma-Aldrich</td>
			<td>P5606</td>
			<td></td>
		</tr>
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			<td>Purina Chow</td>
			<td>Purina</td>
			<td>5001</td>
			<td>Rodent laboratory chow given to rats, mice and hamster is a life-cycle nutrition that has been used in biomedical research for over 5 decades. Provided to rats ad libitum in this experiment.</td>
		</tr>
		<tr>
			<td>Rat cages</td>
			<td>Techniplast</td>
			<td>2000P</td>
			<td>Conventional housing for rodents. Cages were used for housing rats throughout the experiment</td>
		</tr>
		<tr>
			<td>Scalpel blades #11</td>
			<td>Sigma-Aldrich</td>
			<td>S2771</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adobe Photoshop CS2 for Windows</td>
			<td>Adobe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ 1.37v</td>
			<td>NIH</td>
			<td></td>
			<td>The source code is freely available. The author, Wayne Rasband (wayne@codon.nih.gov), is at the Research Services Branch, National Institute of Mental Health, Bethesda, Maryland, USA</td>
		</tr>
		<tr>
			<td>Office 365 ProPlus</td>
			<td>Microsoft</td>
			<td>-</td>
			<td>Microsoft Office Excel</td>
		</tr>
		<tr>
			<td>Windows 10</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2,3,5-Triphenyltetrazolium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>298-96-4</td>
			<td></td>
		</tr>
		<tr>
			<td>50% trichloroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>76-03-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96 %</td>
			<td>Romical</td>
			<td></td>
			<td>Flammable liquid</td>
		</tr>
		<tr>
			<td>Evans blue 2%</td>
			<td>Sigma-Aldrich</td>
			<td>314-13-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane, USP 100%</td>
			<td>Piramamal Critical Care, Inc</td>
			<td>NDC 66794-017</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring post-stroke cerebral edema, infarct zone and blood-brain barrier breakdown in a single set of rodent brain samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

Infarct zone measurement
An independent-sample t-test indicated that 19 rats that underwent permanent MCAO demonstrated a significant increase in brain infarct volume compared to the 16 sham-operated rats (MCAO=7.49% &#177; 3.57 vs. Sham = 0.31% &#177; 1.9, t(28.49) = 7.56, p &#60; 0.01 (see Figure 2A)). The data is expressed as a mean percentage of the contralateral hemisphere &#177; SD.
Brain...

Watch this Scientific Journal Video about Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples at JoVE.com

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

In the present protocol, we present a modified middle cerebral artery occlusion technique that measures ischemic brain injury to determinate an infarct zone, brain edema, and BBB permeability in the same set of rodent brains. In terms of ethical and economic costs, the main advantage of this technique is that only one brain sample is required for all three measurements. This assessment of the main parameters of ischemic brain injury will help to comprehensively investigate pathophysiology of ischemic stroke.To begin, remove the rat brain, and cut 2 millimeter thick horizontal sections with a 0.009-inch stainless steel, uncoated, single edge razor blade. Place the sections in 0.005%TTC and incubate them for 30 minutes at 37 degrees Celsius, then place the brain tissue on the microscope slides and perform optical scanning of the six brain slices with a resolution of 1600 x 1600 DPI. Use a photo editor to add a blue filter to the images.After opening the software, go to image, adjustments, and channel mixer. Select blue as the output channel and check monochrome, then click okay. Save the image in the JPEG file format.Use the polygon selection tool in ImageJ version 1.37 to select each hemisphere of each of the six brain slices and save it as a separate image file. To calculate the infarct volume and brain edema, use a macro to convert the pixels in the image to white or black, based on a threshold. Then, close all files and use a second macro to compare the number of white and black pixels to determine the infarct volume and brain edema.Copy the raw data to a spreadsheet. When calculating infarct volume, correct for tissue swelling by using the ratios of ipsilateral and contralateral cerebral hemispheres. Express the brain edema areas as a percentage of the standard areas of the unaffected contralateral hemisphere.Determine blood brain barrier disruption 24 hours after occlusion. Divide the right and left hemispheres into six slices and put each slice into a microcentrifuge tube. Homogenize each slice of the brain tissue in trichloroacetic acid, using 4 milliliters of 50%trichloroacetic acid per gram of brain tissue.Centrifuge the samples at 10, 000 times G for 20 minutes, then transfer the supernatant into a fresh tube and dilute it 1:3 with 96%ethanol. Perform luminescence spectrophotometry with a plate reader. An independent sample t-test indicated that 19 rats that underwent permanent middle cerebral artery occlusion, or MCAO, demonstrated a significant increase in brain infarct volume compared to the 16 sham operated rats.The rats that underwent permanent MCAO also demonstrated a significant increase in the extent of brain edema and blood brain barrier breakdown after 24 hours, compared to the sham operated animals. When attempting this procedure, it is important to ensure that chemical concentration comply with the protocol. This protocol makes it possible to evaluate all three parameters of ischemic damage in one sample of the brain.

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